The present gold standard for measuring the affinity between two (bio)molecules (called ligands) is surface plasmon resonance (SPR) [see Schasfoort, R. B. M., Tudos, A. J., Handbook of Surface Plasmon Resonance; Royal Society of Chemistry: Cambridge, U.K. (2008)]. One of the ligands is bound to a surface layer (the “bait” layer) which is mounted in a microfluidics device. The other ligand (the “prey”) is injected as a square concentration pulse at different concentrations. Association/dissociation rates, and binding constants, can be calculated from the output signal after application of an adsorption binding kinetics model. The quartz microbalance (QMB, see Liu, Y., Jaiswal, A., Poggi, M. A., Wilson, W. D., Surface Plasmon Resonance and Quartz Crystal Microbalance Methods for Detection of Molecular Interactions; and in Chemosensors: Principles, Strategies, and Applications; Wang, B., Anslyn, E. V., Eds.; John Wiley & Sons Inc.: Hoboken, N.J., 2011; Chapter 16), evanescent wave sensors (see Strehlitz, B.; Nikolaus, N.; Stoltenburg, R., Sensors 2008, 8, 4296-4307) and affinity chromatographic systems [see Tong, Z. H.; Schiel, J. E.; Papastavros, E.; Ohnmacht, C. M.; Smith, Q. R.; Hage, D. S., J. Chromatogr. A 1218, 2011, 2065-2071] are also able to yield quantitative information on the affinity between ligands (see Nienhaus, G. U. Protein-Ligand Interactions: Methods And Applications; Humana Press: Totowa, N.J., 2005). These techniques, which are still in full scientific development, do not require fluorescent labeling (label-free) or extensive sample cleanup. They can be used in fluidic environments.
Potentiometric techniques rely on the development of a potential over a membrane. These potentials are often created by adsorption/diffusion phenomena taking place at the sensor surface. A recent expert update of ideas in the discussion of how these potentials are created is given by Lewenstam [see Lewenstam, A. J., Solid State Electrochem. 15 (2011) 15-22]. Changes of potentials at all kinds of surfaces are well-known in nature's colloidal systems and biomembrane structures. In analytical laboratories potentiometry is mostly performed with ion-selective electrodes (ISEs). The market for such sensors is very extensive, and especially for biomedical and microbiological research, they are part of highly automated equipment with high sample throughput. Several groups have shown that it is possible to use them for targeting charged organic molecules [see Bakker, E.; Pretsch, E. Angew. Chem., Int. Ed. 46 (2007) 5660-5668; and Stefan-van Staden, R. I.; van Staden, J. F.; Aboul-Enein, H. Y. Anal. Bioanal. Chem. 394 (2009) 821-826], multiply charged polymeric and oligomeric substances [see Fu, B.; Bakker, E.; Yun, J. H.; Wang, E.; Yang, V. C.; Meyerhoff, M. E., Electroanalysis 7 (1995) 823-829; and Nagels, L. J.; Everaert, J.; Bohets, H.; Del Favero, J.; Goossens, D.; Robbens, J.; Pietraszkiewicz, M.; Pietraszkiewicz, O. Comb. Chem. High Throughput Screening 10 (2007) 555-559], and even bioparticles [see Tang, D. P.; Yuan, R.; Chai, Y.; Fu, Y.; Dai, J.; Liu, Y.; Zhong, X., Biosens. Bioelectron. 21 (2005) 539-548].
There is a need for an alternative more accessible technique for studying the molecular interactions of (bio)molecules.